1. Field of the Invention
The present invention relates to crystals of the (2R,4R) stereoisomer of monatin and the use thereof. More particularly, the present invention relates to crystals of free (2R,4R)-monatin, i.e., (2R,4R)-monatin in the free (non-salt) form, which is a non-naturally occurring stereoisomer of the naturally occurring (2S,4S) stereoisomer, and which is excellent as a sweetening agent or an active ingredient (sweetener) thereof. The present invention further relates to crystals of a mixture of free (2R,4R)-monatin with at least one other type of monatin stereoisomer and the use of such crystals. The present invention particularly relates to crystals of a mixture of free (2R,4R)-monatin with at least one other type of monatin stereoisomer, such as (2S,4S)-monatin, (2S,4R)-monatin, and (2R,4S)-monatin. The present invention does not relate to a crystal of a mixture of free (2S,4S)-monatin and free (2R,4R)-monatin in a molar ratio of 1:1.
2. Discussion of the Background
As a result of modern eating habits, obesity, arising from the excessive ingestion of sugars, and the various diseases accompanied thereby have become problems of medical and social importance. Accordingly, there has been a strong demand for the development of a low calorie sweetener to replace sugar. Many various properties and requirements, such as low calorie content, safety, stability to heat and acid, sweetness quality, and cost, in addition to sweetness intensity, have been demanded for such sweetening agents.
At present, various kinds of sweetening agents have been used or proposed. For example, aspartame, which is excellent in safety and sweetness quality, and has a strong sweetness intensity (degree of sweetness), has come into practical use as a sweetening agent capable of industrial mass production and has been widely used. Thaumatin, glycyrrhizin, stevioside, and the like, which exist naturally, can be collected in large amounts and are derived from plants, are currently used as natural sweetening agents. Under such circumstances, development of a sweet substance, which has a strong degree of sweetness, for practical use as a sweetening agent has been required.
Monatin is a naturally occurring amino acid derivative isolated from the bark of the roots of Schlerochiton ilicifolius, which is a plant naturally grown in the area of the north-western Transvaal of South Africa. The structure of monatin has been reported as (2S,4S)-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl)-pentanoic acid[((2S,2S)-4-hydroxy-4-(3-indolylmethyl))-glutamic acid (see, Vleggaar, R. et al., J. Chem. Soc. Perkin Trans., pp. 3095-3098 (1992) (Vleggaar, R. et al.) and refer to the structural formula (1) described below for its structure). Vleggaar et al. reported the sweetness intensity of this (2S,4S) substance (natural type monatin) derived from the natural plant to be 800 to 1400 times that of sucrose.
The information for monatin is summarized as follows.
Although several synthetic methods have been reported for producing monatin, many of them afford a mixture of stereoisomers. There has been nearly no report where each of the four stereoisomers having the same chemical structural formula as that of natural monatin is synthesized and isolated as a pure substance and the properties thereof investigated in detail. For synthetic examples of monatin, see: (1) Republic of South Africa Patent Application No. 87/4288 (van Wyk, P. J. et al., ZA 87/4288); (2) Republic of South Africa Patent Application No. 88/4220 (van Wyk. P. J. et al., ZA 84/4220); (3) U.S. Pat. No. 5,994,559 (Abushanab, E. et al., U.S. Pat. No. 5,994,559 (1999)); (4) Holzapfel et al., Synthetic Communications, vol. 24 (22), pp. 3197-3211 (1994) (Holzapfel et al.); and (5) Nakamura, K. et al., Organic Letters, vol. 2, pp. 2967-2970 (2000) (Nakamura, K. et al.).
As for the relationships of the stereochemistry and the sweetness intensity of monatin, van Wyk, P. J. et al., ZA 87/4288 and van Wyk, P. J. et al., ZA 84/4220, in which this issue was noted for the first time, reported that the stereoisomer present in nature and having a strong sweet taste is the (2S,4S) substance or the (2R,4R) substance based on an X-ray crystal structure analysis. It was also reported that there is a high probability that the stereoisomer present in nature and having a strong sweet taste is the (2S,4S) substance, based on the results of the synthesis of a mixture of the (2S,4S) substance and the (2S,4R) substance from (2S)-aspartic acid. Vleggaar, R. et al., mentioned above, has reported that (2S,4S)-monatin is the only stereoisomer (stereo structure) of monatin present in natural plants and its sweetness intensity is 800 to 1400 times that of sucrose. Based on this information, it was reasonable to conclude that the naturally occurring monatin stereoisomer (stereo structure) which exhibits a strong sweetness is the (2S,4S) substance.
Nakamura, K. et al., cited above, reported the isolation of hydrochloride salts of (2S,4S)-monatin and of (2S,4R)-monatin, and that, with regard to the intensity of sweet taste thereof, the synthetic (2S,4S)-monatin exhibited a sweetness potency equivalent to that of the natural specimen of monatin (the (2S,4S) substance) and that the synthetic (2S,4R)-monatin exhibits a slightly sweet taste presumably due to (2S,4S)-monatin, which is thought to be present as an impurity. However, the specific intensity of the sweet taste for (2S,4S)- and (2S,4R)-monatin was not reported. Thus, this reference is the first case in which the sweetness intensity of a non-naturally occurring stereoisomer (other than (2S,4S)) of monatin was noted, but it was reported that there was almost no sweetness in the hydrochloride salt of (2S,4R)-monatin.
Meanwhile, selective synthetic methods for the respective stereoisomers of monatin as sweeteners have been reported (see, Kitahara, T. et al., Annual Meeting in 2000, Abstracts of Papers, 3B128β (page 221), Japan Society for Bioscience, Biotechnology and Agrochemistry (Kitahara, T. et al.), but the degree of sweetness of each stereoisomer has not been reported.
Thus, with respect to the relationships between the stereochemistry and the sweetness intensity of monatin, the following has been found:
1) The naturally occurring monatin stereoisomer (stereo structure) with a sweetness intensity of 800 to 1400 times that of sucrose is the (2S,4S) substance; and
2) Although small amounts of a portion of the other non-naturally occurring stereoisomers of monatin have been isolated, there is no example in which a pure substance has been isolated, purified, and the intensity of its sweetness assessed.
That is, up to the present, with respect to the sweetness intensity of each isomer of monatin at a practical use concentration corresponding to a sucrose concentration of 5 to 10%, there has been no definite data except those of the naturally occurring monatin, i.e., the (2S,4S) substance. Therefore, it could not have been known from the existing references and patent literature references whether the non-naturally occurring stereoisomers of monatin other than (2S,4S)-monatin are usable as sweetening agents. In other words, except for the naturally occurring (2S,4S)-monatin, reliable information for the degree of sweetness (sweetness intensity) has been poor, and thus, in consideration of the totality of the prior art, one could not help but think that the non-naturally occurring monatin isomers, i.e., those other than (2S,4S)-monatin, would have a low degree of sweetness and would not be expected to be useful as sweeteners.
One reason for this conclusion is that a method for synthesizing and isolating/purifying the above various isomers has not been found. Specifically, to assess the utility of (2S,4S)-monatin and the stereoisomers thereof (i.e., the non-naturally occurring stereoisomers) as sweetening agents and to consequently develop a useful sweetening agent comprising such an ingredient, it is necessary to isolate and purify at least several hundred milligrams of naturally occurring monatin as well as the three non-naturally occurring stereoisomers thereof as purified compounds and to study the optical purity and sweetness intensity of those compounds.
To accomplish the above purpose, a method for separating and obtaining the various isomers of monatin in high purity is necessary, and as one possible method, there is the method of crystallization. Based on a search of prior patents and references, the information on crystals of monatin (including forms of free compounds, salts, and the like) is as follows.
In Vleggaar, R. et al., mentioned above, it has been reported that crystals of the free compound of monatin, the (2S,4S) substance, are obtained from a mixed solvent of water, acetic acid, and ethanol (1:1:5), and have a melting point of from 216° C. to 220° C. Also, in van Wyk, P. J. et al., ZA 87/4288 and van Wyk, P. J. et al., ZA 84/4220, it has been described that the melting point of the free compound of monatin, the (2S,4S) substance, (crystalline solid) is from 247° C. to 265° C. (decomposition), but it has also been reported that various salts are amorphous solids. In Holzapfel, C. W. et al., mentioned above, it has been reported that crystals of free compound of a mixture of synthetic (2S,4S)-monatin and (2R,4R)-monatin are obtained from a mixed solvent of water and acetic acid (10:1) by crystallizing twice, and that its melting point is from 212° C. to 214° C.
Therefore, except for these three cases with respect to the free compound of the (2S,4S)-monatin substance, concerning non-naturally occurring stereoisomers of monatin and mixtures of those multiple stereoisomers there of, free compounds thereof as a matter of course and various salts thereof have not been isolated in the crystalline state. Therefore, the physical properties and other information are not known at all. That is, with respect to monatin, there is no report of a crystallization method (which is the simplest and most effective method for purification compared with conventional purification methods such as an ion exchange chromatography) or findings for crystals obtained therefrom, other than only two cases for a free form compound of (2S,4S)-monatin and a mixture of free form compounds of (2S,4S)-monatin and (2R,4R)-monatin, and, in particular, no findings have been reported for crystals of salts.
Taking into account the above information, the present inventors separated four stereoisomers of monatin, and evaluated the degree of sweetness of the sodium salt of each stereoisomer using a 5% sucrose solution as a control. As a result, it has been demonstrated that the degree of sweetness is 50 times for a (2S,4S)-monatin substance, 300 times for a (2S,4R)-monatin substance, 2700 times for a (2R,4R)-monatin substance, and 1300 times for a (2R,4S)-monatin substance, and that (2R) monatin substances are excellent in the degree of sweetness and useful as a sweetening agent (see, PCT International Application Publication No. 03/045914 (Amino, Y. et al., WO 2003045914 A1)). In Amino, Y. et al., WO 2003045914 A1, a salt crystal of each stereoisomer was obtained as a novel substance, and was found to be excellent as a sweetening agent in terms of being thoroughly soluble in water and easily separated/purified. Furthermore, it has been found that such a salt crystal has superiority in terms of storage stability under high temperature conditions as a crystal, compared to a crystal of free (2R,4R)-monatin prepared according to the preparation method of a crystal of free (2S,4S)-monatin publicly known in Vleggaar, R. et al., noted above (see, Amino, Y. et al., WO 2003045914 A1).
In this way, the inventors prepared salt crystals of the monatin stereoisomers as novel substances, and showed the usefulness thereof in Amino, Y. et al., WO 2003045914 A1. At that time, in order to compare the natures of the various salt crystals to those of the crystals of the free compounds, crystals of free (2R,4R)-monatin were prepared according to the method described in Vleggaar, R. et al., noted above (see, Comparative Example 1 of Amino, Y. et al., WO 2003045914 A1). Since this crystal of free (2R,4R)-monatin is an enantiomer of the crystal of free (2S,4S)-monatin described in Vleggaar, R. et al., they were expected to have the same physical properties. However, the crystal of free (2R,4R)-monatin prepared by the inventors obviously had different physical properties from those of the crystal of free (2S,4S)-monatin cited in Vleggaar, R. et al., and raised the possibility that it is a novel crystal (form). That is, the melting point of the crystal of free (2R,4R)-monatin, which should have the same value as that of the crystal of free (2S,4S)-monatin described in Vleggaar, R. et al., was 175.2 to 176.1° C., which was far from the melting point of the crystal of free (2S,4S)-monatin described in Vleggaar, R. et al., 216 to 220° C. As described below, remeasuring the melting point of the crystal of free (2R,4R) monatin substance gave a value of from 171.5 to 172.7° C., which nearly reproduced the value in Comparative Example 1 of Amino, Y. et al., WO 2003045914 A1.
Therefore, it was desired to examine the preparation of crystals of free (2R,4R)-monatin and the resultant crystals of free (2R,4R)-monatin in further detail, and clarify whether this is a different crystal or crystal form from the crystal of free (2S,4S)-monatin reported in Vleggaar, R. et al., as well as to investigate the usefulness thereof as a sweetening agent.